Transcriptome analysis of bull spermatozoa: implications for male fertility.

Spermatozoa deliver more than the paternal genome into the oocyte; they also carry remnant messenger RNA from spermatogenesis. The RNA profiles of spermatozoa from high-fertility and a low-fertility Holstein bulls were analysed using Affymetrix bovine genechips. A total of 415 transcripts out of approximately 24,000 were differentially detected in spermatozoa collected from both bulls (fold change > or =2.0; P<0.01). These transcripts were associated with different cellular functions and biological processes. Spermatozoa from high-fertility bulls contained higher concentrations of transcripts for membrane and extracellular space protein locations, while spermatozoa from the low-fertility bulls were deficient of transcripts for transcriptional and translational factors. Quantitative real-time PCR was used on three low-fertility and four high-fertility bulls to validate the microarray data. Two highly represented transcripts in the microarray analysis (protamine 1 and casein beta 2) were validated, as well as a third transcript (thrombospondin receptor CD36 molecule) that showed a lower concentration in low-fertility bulls. This study presents the global analysis of spermatozoa originating from bulls with opposite fertility. These results provide some specific transcripts in spermatozoa that could be associated with bull fertility.

Spermatozoal RNA: Why is it there and what does it do?

The mammalian male gamete is transcriptionally silent as a consequence of the highly condensed architecture of its chromatin and there is also little or no cytoplasm capable of supporting translation; however, we now understand that these cells carry a full complement of mRNAs and that under certain conditions, spermatozoa can translate their mRNAs de novo. Moreover, there is now good evidence that spermatozoal RNA can potentially affect phenotypic traits in offspring. This epigenetic phenomenon may involve the transmission of extra-chromosomal episomal elements. These data indicate that the spermatozoon is well matched to its role of delivering the paternal genome, but that additional male-benefits may also be accrued by the delivery of spermatozoal RNA.

Measurement of bovine sperm nuclear shape using Fourier harmonic amplitudes.

An objective method for measuring bovine sperm nuclear shape was developed. Digital images of bovine sperm stained with propidium iodide were collected and Fourier functions used to describe the perimeters of individual sperm nuclei. Harmonic amplitudes from Fourier functions were first shown to be independent of sperm orientation during digitization. Sperm from 12 different bulls were used, and 6 harmonic amplitudes per sperm were found to adequately describe sperm nuclear shape. Based on harmonic amplitudes 0 to 5, cluster analysis was used to generate 20 different groups. Sperm within groups had similar morphologies and groups were distinguished by statistically unique shape characteristics. Harmonic amplitudes 0 to 5 can be used to distinguish previously reported abnormalities such as tapered, pyriform, macrocephalic, and microcephalic, as well as gradations in between. Furthermore, differences were detected among bull harmonic amplitude centroids (P < .05), indicating that bulls differ in mean sperm nuclear shape.

Relationship of bull fertility to sperm nuclear shape.

The relationship between sperm nuclear shape and bull fertility was determined. Two groups of bulls, 3 per group, were selected. Bulls differed in fertility based on lifetime nonreturn rates. Digital images of propidium iodide-stained sperm from each bull were collected and shape-evaluated by Fourier harmonic amplitudes 0 to 5. A discriminant function (P < .05) was constructed based on harmonic amplitudes and the 2 fertility groups. When individual sperm were classified as being of high or lower fertility, the percentage of each bull's sperm placed in the high-fertility group had a linear relationship (r = .89, P < .05) with fertility. To construct a plot of mean sperm shapes, a novel technique to automatically orient and identify the anterior tip of the sperm head was developed. The mean nuclear shape of high-fertility sperm was more elongated and tapered than those of lower fertility. A discriminant function (P < .05) was also constructed that separated the 6 bulls into 2 groups based only on the harmonic amplitudes or sperm nuclear shape. The bulls were correctly classified into the 2 fertility groups. A comparison of sperm chromatin structure analysis (SCSA) and harmonic amplitudes found that overall size variance, anterior roundness, and posterior taperedness of sperm nuclei were related to chromatin stability (P < .05). Some of the differences observed in sperm nuclear shape between the high- and lower-fertility bulls may be explained by varying levels of chromatin stability. However, sperm nuclear shape appears to contain additional information from chromatin stability alone. In this particular study, with 6 bulls, all with good chromatin quality, sperm nuclear shape was a better predictor of bull fertility.